In many clinical approaches of gene therapy until now, viral vectors from retroviruses, adenoviruses, and adeno-associated viruses have been used. These gene therapy vectors have limitations in gene introducing efficiency and persistent expression, and also have cell toxicity, and immunogenicity, which are crucial problems when it comes to the medical application of these vectors (Lamb, R. A. & Kolakofsky, D., Paramyxoviridae: the viruses and their replication in Fields Virology, 3rd edn, (Edited by B. N. Fields, D. M. Knipe & P. P. Howley) pp. 1177–1204 (Philadelphia, Lippincott-Raven (1996)). Novel vectors based on lentiviruses and HSV have been proposed as countermeasures, and extensive research is also being carried out to improve existing vectors. However, all of these vectors exist in the form of DNA within the nucleus throughout the life cycle. Therefore, it is difficult to fully overcome concerns of safety related to random interactions with the patient's chromosomes.
Recent rapid progress of reverse genetics technologies is making it possible to develop vectors based on RNA viruses, the development of which has been long delayed. Recombinant RNA virus vectors show high gene introduction efficiency and expression capability, and thus show a very high potentiality as vectors for gene therapy (Roberts, A. & Rose, J. K., Virology 247, 1–6 (1998); Rose, J., Proc. Natl. Acad. Sci. USA 94, 14998–15000 (1996); Palese, P. et al., Proc. Natl. Acad. Sci. USA 93, 11354–11358 (1996)). However, practically usable paramyxovirus vectors derived from deficient type genome of attenuated viruses have not been reported yet.
Paramyxovirus vectors having negative-strand RNA as the genome have several characteristics significantly different from retroviruses, DNA viruses or positive-strand RNA virus vectors. Genomes or antigenomes of negative-strand RNA viruses do not directly function as mRNA, so they cannot initiate the synthesis of viral proteins and genome replication. Both RNA genome and antigenome of these viruses always exist in the form of a ribonucleoprotein complex (RNP), so they hardly cause problems caused by antisense strands, such as interfering with the assembly of genome to RNP due to mRNA hybridizing with naked genomic RNA, as in the case of positive strand RNA viruses. These viruses comprise their own RNA polymerases, performing the transcription of viral mRNAs or replication of viral genomes using RNP complex as the template. Worthy of mentioning is that negative-strand RNA (nsRNA) viruses proliferate only in the cytoplasm of host cells, causing no integration thereof into chromosomes, because they do not go through a DNA phase. Furthermore, no homologous recombination among RNAs has been recognized. These properties are considered to contribute a great deal to the stability and safety of negative-strand RNA viruses as gene expressing vectors.
Among negative-strand RNA viruses, the present inventors have been focusing their attention on the Sendai virus (SeV). Sendai virus is a non-segmented type negative-strand RNA virus belonging to the genus Paramyxovirus, and is a type of murine parainfluenza virus. The virus attaches to the host cell membrane via envelope glycoproteins, the hemagglutinin-neuramimidase (HN) and fusion protein (F), causes membrane fusion, and efficiently releases its own RNA polymerase and the RNA genome, which exists as a ribonucleoprotein (RNP) complex, into the cytoplasm, and carries out mRNA transcription of the virus and genome replication at the site (Bitzer, M. et al., J. Virol. 71(7):5481–5486, 1997). The viral envelope protein F is synthesized as an inactive precursor protein (F0), then divided into F1 and F2 by proteolytic cleavage with trypsin-like protease such as triptase clara (Kido, H. et al., Biopolymers (Peptide Science) 51(1): 79–86, 1999), and thus becomes an active form protein to cause membrane fusion. This virus has been said to be non-pathogenic towards humans. However, wild-type SeV has been said highly cytopathic in cell culture (D. Garcin, G. Taylor, K. Tanebayashi, R. Compans and D. Kolakofsky, Virology 243, 340–353 (1998)). Therefore, we focused research on Z strain of SeV, an attenuated laboratory strain of Sendai virus, which has been isolated, and which only induces mild pneumonia in rodents, the natural hosts. This strain has been widely used as a research model for molecular level studies of the transcription-replication mechanism of paramyxoviruses and used for preparing hybridomas. In addition to the high safety mentioned above, the virus shows a high production titer of 109-11 pfu/ml in cell lines or chicken eggs. In one recently successful recovery system of negative-strand RNA virus vector from cDNA, especially high reconstitution efficiency has been seen in the case of Sendai virus. The capability of recombinant wild type viruses introduced with exogenous genes, to efficiently and stably express introduced exogenous genes is gaining wide attention.
Thus, negative-strand RNA viruses have many advantages as gene introducing vectors. However, to apply for gene therapy, the development of highly safe vectors that do not release infectious particles when infected to cells is desired. For that purpose, a technique that mass produces viruses deficient in wild type virus production capability is necessary. However, development of an applicable vector based on an envelope gene-deficient genome has not yet been successful.